Recent evidence indicates that the amplification of genes which encode the target enzymes of various antiproliferative agents can mediate resistance to these substances in eukaryotic cells. Studies are proposed to enhance our understanding of the molecular structure of units of gene amplification, eukaryotic structural genes, and the mchanism(s) mediating gene amplification. The amplification of the genes encoding the first three enzymes of uridine biosynthesis (CAD gene) will be studied in cells resistant to N-(phosphonacetyl)-L-aspartate (PALA). One approach will use the technique of in vitro gene transfer to examine whether gene location affects the structure of the amplified unit. CAD genes obtained from physically or enzymatically cleaved chromosmes will be inserted into the genomes of cells lacking this gene, and transformants will be detected by their ability to grow in the absence of uridine. These clones will be selected for resistance to PALA and the location, size, and structure of their amplified units will be investigated. CAD genes constructed in vitro using recombinant DNA technology will be analyzed for function using in vitro gene transfer as a bioassay.